1. Field of the Invention
The present invention relates to a novel peptide having osteogenetic activity and an osteogenetic accelerator containing the same as an active ingredient.
The peptide of the present invention, which has the osteogenetic activity, is useful for treatment of fractures, as a filler in deficient sites of bone, for inhibition of decrease in bone substance related to osteoporosis and periodontic diseases, for prevention of fractures associated with osteoporosis and rheumatoid arthritis and the like.
2. Description of Related Art
Bone morphogenetic protein (BMP) is a member of transforming growth factor (TGF) xcex2 family (Wozney, J. M. et al, Science, 242, 1528 (1988)), and its active form exists as a homodimer having a molecular weight of about 18kD. BMP has the function of acting on undifferentiated mesenchymal cells, inducing differentiation to chondroblasts and osteoblasts and effecting chondrogenesis and osteogenesis (Wang, E. A. et al. Proc. Natl. Acad. Sci. USA, 87, 2220 (1990)).
For this reason, BMP is expected to be effective in treatment of fractures, inhibition of decrease in the bone substance related to osteoporosis and periodontic diseases, in prevention of fractures associated with osteoporosis and rheumatoid arthritis and the like (for example, see Japanese Unexamined Patent Publications Nos.
HEI 6(1994)-298800 and HEI 10(1998)-70989).
Also, there are known a number of inventions relating to implants and compositions in which BMP is combined with a variety of matrices (for example, see Japanese Unexamined Patent Publications Nos. HEI 6(1994)-296677, HEI7(1995)-246235, HEI 7(1995)-116240, HEI 7(1995)-88174 and HEI 10(1998)-151188).
However, the above-described BMP, when it is administered in vivo, disappears from blood within a few minutes and loses its effect. If administered in a large amount for compensating that, BMP might possibly cause various adverse effects, including toxic effects on livers and kidneys. Further, BMP has an immunogenicity because of its large molecular weight, and might possibly cause anaphylactic shock when administered repeatedly. furthermore, where BMP is impregnated in matrices of decalcificated bone or collagen for use, osteogenetic activity is expressed, but there may be another problem of antigenicity or infection attributed to the matrices.
Under these circumstances, an object of the present invention is to provide a peptide having an osteogenetic activity in which peptide the above-mentioned problems are alleviated, and an osteogenetic accelerator containing the peptide.
According to the present invention, the above-mentioned object is achieved by a peptide having any one of the sequences SEQ ID NO.1 to SEQ ID NO.8.
Also the object of the present invention is achieved by an osteogenetic accelerator containing the above-mentioned peptide.
These and other objects of the present application will become more readily apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The peptides of the present invention are not necessarily required to have exactly the same amino acid sequence as represented by any one of SEQ ID NO.1 to SEQ ID NO.8, provided that they have the osteogenetic activity. In other words, so long as the peptides have the osteogenetic activity, one to several amino acids in the sequences may optionally be deleted or substituted or one to several amino acids may optionally be added to the sequences, by a usual technique in genetic engineering or in peptide synthesis. The optionally deleted, substituted or added amino acids may be selected as appropriate depending on the kind of amino acids, a site and the like.
In the present invention, to xe2x80x9chave the osteogenetic activityxe2x80x9d may be construed as activity of accelerating the activation of alkaline phosphatase in osteoblasts (Yamaguchi,A., Molecular Medicine, Vol.30, No.10, 1232(1993)) so as to form neogenetic bone or induce growth of existing bone.
In the present specification, amino acid residues are represented by abbreviatory symbols as follows:
Ala: L-alanine residue
Asn: L-asparagine residue
Cys: L-cysteine residue
Gin: L-glutamine residue
Glu: L-glutamic acid residue
Ile: L-isoleucine residue
Leu: L-leucine residue
Lys: L-lysine residue
Pro: L-proline residue
Ser: L-serine residue
Thr: L-threonine residue
Val: L-valine residue
Glx: L-L-glutamine residue or L-glutamic acid residue
Xaa: amino acid defined in each sequence
Also in the present specification, the amino acid sequence of a peptide is written according to the conventional notation, with an amino group at the N-terminal appearing on the left hand of the sequence and carboxyl group at the C-terminal appearing on the right hand thereof.
The amino acid sequences represented by SEQ ID NO.1 to SEQ ID NO.8 may also be represented by the formula:
xe2x80x94Y1-Asn-Y2-Y3-Y4-Pro-Lys-Y5-Cys-Cys-Y6-Pro-Thr-Y7-Le u-Y8-Ala-Y9xe2x80x94,
wherein Y1 is a peptide residue or an amino acid residue selected from the group consisting of Asn-Ser-Val and Ile, Y2 is an amino acid residue or a peptide residue selected from the group consisting of Ser and Pro-Glu, Y3 is an amino acid residue selected from the group consisting of Lys, Ser and Thr, Y4 is an amino acid residue selected from the group consisting of Ile and Val, Y5 is an amino acid residue selected from the group consisting of Ala and Pro, Y6 is an amino acid residue selected from the group consisting of Ala and Val, Y7 is an amino acid residue selected from the group consisting of Glu and Gln, Y8 is an amino acid residue selected from the group consisting of Ser and Asn, Y9 is an amino acid residue or a peptide residue selected from the group consisting of Ile and Ile-Ser.
The peptides of the present invention may be produced by a method usually used for synthesizing peptides, for example, by a solid phase synthesis method or by a liquid phase synthesis method. The solid phase synthesis method is simpler in operation (for example, see xe2x80x9cSequel to Biochemical Experiments 2, Chemistry about Protein (the second volume)xe2x80x9d p.p.641-694 edited by the Biochemical Society in Japan published on May 20, 1987 by Tokyo Kagaku Dojin, Japan and xe2x80x9cSolid Phase Peptide Synthesisxe2x80x94A Practical Methodxe2x80x9d p.p.152-154by Atherton, E. et al. published in 1989 by IRL Press, Oxford). The solid phase synthesis can be carried out usually by protecting amino groups with appropriate protecting groups, for example, either Boc (tert-butoxycarbonyl) or Fmoc (9-fluorenylmethyloxycarbonyl), or a combination thereof.
For producing the peptide of the present invention, for example, 1) an amino acid corresponding to the C-terminal of the peptide to be produced is bonded to a solid phase material insoluble to a reaction solvent via an xcex1-COOH group of the amino acid; 2) subsequently, in the direction to the N-terminal of the peptide, a corresponding amino acid or peptide fragment is bonded by condensation to the amino acid of 1) after protecting other functional groups such as an xcex1-amino group of the corresponding amino acid or peptide fragment other than an xcex1-COOH group; 3) a protecting group of an amino group forming a peptide bond such as an xcex1-amino group is removed from the bonded amino acid or peptide fragment; these steps are repeated to elongate a peptide chain in order to form a peptide chain corresponding to the desired peptide.
The thus produced peptide chain is detached from the solid phase material and protecting groups are removed from protected functional groups. Subsequently the peptide chain is purified, thereby to obtain the desired peptide.
Here, as the solid phase material, styrene-divinyl benzene copolymers, Merrifield resins, chloromethyl resins, Wang resins, Sieber resins, rink amide resins, rink acid resins, 2-chlorotrityl chloride resins, HMBA-MBHA resins, MBHA resins, oxime resins and the like may be used. Among these resins, styrene-divinyl benzene copolymers are preferred.
It is preferred from the viewpoint of preventing side reaction that the detachment of the peptide chain from the solid phase material and the removal of the protecting groups are carried out simultaneously using trifluoroacetic acid or hydrogen fluoride.
As a solvent and a condensing agent in the peptide synthesis, any ones usually known in the art may be used as required. For example, DMF (dimethylformamide), trichloroethanol, N-methylpyrrolidone and the like may be mentioned as solvents, and DCC, HATU (0-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate), HOBt (1-hydroxybenzotriazole), HBTU (0-benzotriazole-1-yl-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl uronium hexafluorophosphate), PyBOP (benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate), CF3-NO2-PyBOP and the like may be mentioned as condensing agents.
For purifying the obtained peptide, it is effective to utilize reverse phase liquid chromatography.
Either or both of the N- and C-terminals of the peptide of the present invention may optionally be modified chemically. For example, the N-terminal may be acetylated and the C-terminal may be amidated.
The peptide of the present invention may form a physiologically acceptable salt by conventional salt formation reaction. Such salts can include salts with inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid; salts with organic acids such as lactic acid, tartaric acid, maleic acid, fumaric acid, oxalic acid, malic acid, citric acid, oleic acid and palmitic acid; salts with hydroxides and carbonates of alkali metals and alkali earth metals such as sodium, potassium, calcium and aluminum; and salts with amines such as triethylamine, benzylamine, diethanolamine, t-butylamine, dicyclohexylamine and arginine.
Preferred examples of peptides provided by the present invention are peptides having amino acid sequences represented by SEQ ID NO.9 or SEQ ID NO.10, which are examples of SEQ ID NO.1 and peptides having an amino acid sequence represented by SEQ ID NO. 11, which is an example of SEQ ID NO.8, and particularly peptides having amino acid sequences represented by SEQ ID NO. 9 to SEQ ID NO.11 and having an amino group at the N-terminal and a carboxyl group at the C-terminal, and peptides having an amino acid sequence represented by SEQ ID NO.9 and having an acetyl group at the N-terminal and a carboxyl group at the C-terminal.
These peptides may have a part of thereof deleted or substituted or have addition to amino acid(s) thereto so long as they have the osteogenetic activity, as described above.
It is verified that the peptide of the present invention has the osteogenetic activity and is negligible in toxicity such as cytotoxity, systemic acute toxicity and the like. As for the osteogenetic accelerator comprised of the peptide of the present invention, it is possible to avoid the problems of infection and antigenicity resulting from matrices by means of sterilizing operation such as xcex3-ray sterilization, moist heat vapor sterilization and selection of a carrier made of a polysaccharide having low antigenicity or the like during production process.
The peptide of the present invention may be used singly for the purpose of preventing or treating bone fractures. Also the peptide may be used in the form of an osteogenetic accelerator obtained by fixing, mixing, solving or suspending the peptide in a proper carrier or an aqueous solvent which can contain a variety of pharmacologically acceptable additives such as a stabilizer, a preservative, a thickener, a solubilizer and the like. It is particularly preferable that the osteogenetic accelerator of the present invention is one in which the peptide is fixed to a carrier.
The carrier for fixing the peptide of the present invention is not particularly limited to any type provided that it has compatibility to living bodies. For example, it is possible to use, singly or in combination, carriers which can be degraded and absorbed in vivo, such as covalently crosslinked gels of alginate (Suzuki,Y. et al., J. Biomed. Mater. Res., 39, 317(1998)), gels of protein such as collagen, hyaluronic acid, calcium sulfate, polylactic acid, polyglycolic acid, hydroxyapatite, tricalcium phosphate and the like, as well as various ceramics and artificial bone. In addition, starch gel, chitin/chitosan gel, agarose gel and dextran gel may be used as polysaccharide gels. Among these carriers, a covalently crosslinked gel of alginate and a gel of hyaluronic acid are preferred from the view point of non-inflammatory (J.Biomed.Mater.Res.Appl.Biomater., 48, 522-527 (1999) and J.Artif.Organs, 1, 28-32 (1998)) and non-immunogenic (J.Biomed.Master.Res., 1994, Sep;28(9):1037-46) properties.
The method of fixing the peptide on a carrier is not particularly limited. It is possible to adopt a fixation method allowing formation of covalent bond, ionic bond, hydrophobic bond, hydrogen bond, SS bond or the like, for example, an immersion, impregnation, spray, application and dropping method with use of a solution containing the peptide. Among these fixation methods, fixation by covalent bond is preferred from the viewpoint of stability and continuity of effect. Such fixation can be done by a method usually used for fixing a physiologically active protein such as an enzyme (for example, see Scouten, W. H., Methods in Enzymol., 135, Mosbach, K. Ed., 1987, Academic Press NY, p.p.30-65).
Preferably, the peptide to be fixed is used in an amount of about 0.01 to about 50 parts by weight, preferably about 0.1 to 25 parts by weight, with respect to 100 parts by weight of a dry carrier. The peptide thus fixed is usually used for treatment of a fracture or the like by being implanted in a deficient site in bone. If the peptide is used in an amount smaller than 0.01 parts by weight with respect to 100 parts by weight of a dry carrier, the effect of the peptide tends to be insufficient. If the peptide is used in an amount larger than 50 parts by weight, on the other hand, the ratio of fixation of the peptide to the carrier declines and the peptide tends not to be utilized effectively.
As aqueous solvents, physiological saline and physiologically acceptable aqueous solutions of mannitol, sucrose, lactose, maltose, glucose, fructose or the like. A glucose aqueous solution of 5% and a physiological saline are preferable. These aqueous solvents may be used so that the concentration of the peptide is 0.001% to 5%, preferably 0.01% to 1%. If the concentration of the peptide exceeds 5%, the viscosity of the solution rises. Accordingly, there are tendencies that administration becomes difficult and that the peptide separates at an administration site and as a result its effect declines. If the concentration of the peptide is below 0.001%, on the other hand, the effect of the peptide tends to be insufficient.
The osteogenetic accelerator may be used by intravenous, subcutaneous, intraperitoneal, intra-articular or dermal administration or by filling it in a defective site in bone. Further, if capsulated or made into liposomes by the conventional method, the osteogenetic accelerator can be administered orally.
Thus, the peptide and the osteogenetic accelerator of the invention can promote treatment of fractures by being administered to patients with fractures caused by rheumatoid arthritis and osteoporosis or by being filled or implanted in a defective site in bone. Also they can inhibit decrease in bone substance and prevent fractures by being administered to patients with rheumatoid arthritis, osteoporosis and periodontic diseases.
The dose of the peptide as an active ingredient may vary as required depending upon the weight of bone desired to be formed, the site of injured bone, the condition of bone, and the age, sex and weight of a patient and the like. But usually, the peptide expresses its effect by being administered at a dose of 0.01 xcexcg/kg to 33 mg/kg (for an adult), preferably 0.01 xcexcg/kg to 3.3 mg/kg (for an adult), once per day.